Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1410 Laney Walker Blvd, Augusta, GA, 30912, USA.
Georgia Cancer Center, Medical College of Georgia, Augusta, GA, 30912, USA.
BMC Cancer. 2018 Feb 6;18(1):149. doi: 10.1186/s12885-018-4061-y.
Pancreas ductal adenocarcinoma (PDAC) has the most dismal prognosis among all human cancers since it is highly resistant to chemotherapy, radiotherapy and immunotherapy. The anticipated consequence of all therapies is induction of tumor apoptosis. The highly resistance nature of PDACs to all therapies suggests that the intrinsic tumor cell factors, likely the deregulated apoptosis pathway, are key mechanisms underlying PDAC non-response to these therapies, rather than the therapeutic agents themselves. The aim of this study is to test the hypothesis that epigenetic dysregulation of apoptosis mediators underlies PDAC resistance to gemcitabine, the standard chemotherapy for human PDAC.
PDAC cells were analyzed for apoptosis sensitivity in the presence of a selective epigenetic inhibitor. The epigenetic regulation of apoptosis regulators was determined by Western Blotting and quantitative PCR. The specific epigenetic modification of apoptosis regulator promoter chromatin was determined by chromatin immunoprecipitation in PDAC cells.
Inhibition of histone methyltransferase (HMTase) by a selective HMTase inhibitor, verticillin A, significantly increased human PDAC cell sensitivity to gemcitabine-induced growth suppression. Verticillin A treatment decreased FLIP, Mcl-1, Bcl-x and increased Bak, Bax and Bim protein level in the tumor cells, resulting in activation of caspases, elevated cytochrome C release and increased apoptosis as determined by upregulated PARP cleavage in tumor cells. Analysis of human PDAC specimens indicated that the expression levels of anti-apoptotic mediators Bcl-x, Mcl-1, and FLIP were significantly higher, whereas the expression levels of pro-apoptotic mediators Bim, Bak and Bax were dramatically lower in human PDAC tissues as compared to normal pancreas. Verticillin A downregulated H3K4me3 levels at the BCL2L1, CFLAR and MCL-1 promoter to decrease Bcl-x, FLIP and Mcl-1 expression level, and inhibited H3K9me3 levels at the BAK1, BAX and BCL2L11 promoter to upregulate Bak, Bax and Bim expression level.
We determined that PDAC cells use H3K4me3 to activate Bcl-x, FLIP and Mcl-1, and H3K9me3 to silence Bak, Bax and Bim to acquire an apoptosis-resistant phenotype. Therefore, selective inhibition of H3K4me3 and H3K9me3 is potentially an effective approach to overcome PDAC cells resistance to gemcitabine.
胰腺癌（PDAC）是所有人类癌症中预后最差的，因为它对化疗、放疗和免疫治疗具有高度耐药性。所有治疗方法的预期结果都是诱导肿瘤细胞凋亡。PDAC 对所有治疗方法的高度耐药性表明，内在的肿瘤细胞因素，可能是 PDAC 对这些治疗方法无反应的关键机制，而不是治疗药物本身。本研究旨在验证以下假设：即凋亡介质的表观遗传失调是 PDAC 对吉西他滨耐药的基础，吉西他滨是人类 PDAC 的标准化疗药物。
在存在选择性表观遗传抑制剂的情况下，分析 PDAC 细胞的凋亡敏感性。通过 Western Blotting 和定量 PCR 确定凋亡调节剂的表观遗传调控。通过 PDAC 细胞中的染色质免疫沉淀测定凋亡调节剂启动子染色质的特定表观遗传修饰。
选择性组蛋白甲基转移酶（HMTase）抑制剂 verticillin A 抑制 HMTase，可显著增加人 PDAC 细胞对吉西他滨诱导生长抑制的敏感性。Verticillin A 处理降低了肿瘤细胞中的 FLIP、Mcl-1、Bcl-x 水平，增加了 Bak、Bax 和 Bim 蛋白水平，导致 caspase 激活、细胞色素 C 释放增加，并通过肿瘤细胞中 PARP 裂解的上调导致细胞凋亡增加。对人 PDAC 标本的分析表明，与正常胰腺相比，人 PDAC 组织中抗凋亡介质 Bcl-x、Mcl-1 和 FLIP 的表达水平显著升高，而促凋亡介质 Bim、Bak 和 Bax 的表达水平则显著降低。Verticillin A 下调 BCL2L1、CFLAR 和 MCL-1 启动子的 H3K4me3 水平以降低 Bcl-x、FLIP 和 Mcl-1 的表达水平，并抑制 BAK1、BAX 和 BCL2L11 启动子的 H3K9me3 水平以上调 Bak、Bax 和 Bim 的表达水平。
我们确定 PDAC 细胞使用 H3K4me3 激活 Bcl-x、FLIP 和 Mcl-1，使用 H3K9me3 沉默 Bak、Bax 和 Bim，从而获得抗凋亡表型。因此，选择性抑制 H3K4me3 和 H3K9me3 可能是克服 PDAC 细胞对吉西他滨耐药性的有效方法。
